1. Modeled growth of C. protothecoides with Phosphorus and Light as Limiting Nutrients
Patrick Cusack and Rachael Mordovancey
Dr. Caye Drapcho
BE 4100, Biosystems Engineering,
Clemson University, Clemson, SC, 29361
Abstract
This project was performed to model a biological growth system
using the Monod model for multiple limiting substrates. Three
batch reactors inoculated with Chlorella protothecoides were
grown to measure growth in the presence of the plant-based,
phosphorus-free Dropps laundry detergent. The results showed
that C. protothecoides grew best in the absence of the detergent.
Additionally, the Monod model showed that C. protothecoides
would be able to grow exponentially for 2500 hours, after which
the phosphorous concentration would be depleted.
Introduction
In 1948, manufacturers began adding sodium tripolyphosphate
to synthetic detergents for its impressive cleaning abilities. As its
use became more popular, the concentration of phosphorus in
household wastewaters also began to rise. Eventually, this excess
phosphorus wound up in streams, lakes, and the ocean. With
such excess phosphorus, in combination with sufficient sunlight,
nitrogen, and other essential nutrients, freshwater algae
flourished. In some cases, the algae grew too well and depleted
the dissolved oxygen concentration in the water, suffocating fish
and other aquatic animals. When scientists made the connection
between excess phosphorus and eutrophication, most developed
countries banned the use of phosphates in detergents.
Unfortunately, with the widespread use of agricultural
fertilizers, eutrophication is still a prevalent environmental
concern. The purpose of this experiment was to test the effects of
the Dropps laundry detergent on the growth of C. protothecoides
and model the growth of C. protothecoides with phosphorus and
light as the substrates of interest.
Materials and Methods
Three beakers of varying phosphorus levels were inoculated
with 21 mL of C. protothecoides. The first beaker contained 700
mL of BG11 media. The second beaker contained 700 mL of
BG11 media and 540 µL of Dropps laundry detergent. The third
beaker contained 700 mL of deionized water and 540 µL of
Dropps laundry detergent. All three beakers were then placed on
stir plates. The optical density of each sample was measured at
750 nm with a spectrophotometer. The pH of each sample was
measured using a pH reader. Lastly, the phosphorus
concentration of each sample was measured following Method
4500-P D of Standard Methods for the Examination of Water
and Wastewater (APHA, 1995). Additionally, the growth of C.
protothecoides, based on phosphorus concentration and light
intensity, was modeled using the program STELLA.
Conclusions
Although the concentration of C. protothecoides increased and decreased
over time in all three reactors, the algae grew best in the BG11 without the
Dropps laundry detergent. The concentration of phosphorus in each reactor
consistently decreased, indicating phosphorus utilization and biomass
formation. Interestingly, the reactor containing deionized water and the
“phosphorus-free” Dropps detergent supported the growth of C.
protothecoides. Unless there was contamination or errors conducting the
phosphorus test, this indicates that the detergent contains phosphorus.
Unfortunately, with such a short span of time, this data does not accurately
show the effects of the Dropps detergent on C. protothecoides growth.
References
1. Köhler, J. (2016). Detergent Phosphates: An EU Policy Assessment. J. of Business Chem., 3-2.
http://www.businesschemistry.org/article/?article=73
2. Mordovancey, R. 2018: Lab 5 Methods of Biomass Determination: Optical Density and
biomass Dry Weight. BE 4100, Clemson University.
3. Drapcho 2018: Lecture 11 Stoichiometry of Microbial Reactions. Unpublished Notes, BE 4100,
Clemson University.
4. Drapcho 2018: Lecture 12 Modeling Microbial Growth with Monod Model. Unpublished
Notes, BE 4100, Clemson University.
5. Drapcho 2018: Lecture 14 Product Formation and Nutrient Utilization Rates. Unpublished
Notes, BE 4100, Clemson University.
6. SC-Life Presentation Series: How to Create a Poster for a Professional Meeting, Stocks and
Zimmerman, 2005.
Acknowledgements
We would like to thank Dr. Caye Drapcho for her expertise and assistance and the
Environmental Engineering and Earth Sciences department for the equipment and lab
space.
Results
Using the standard curve from a previous lab, shown below in Figure 1, the
concentration of C. protothecoides in each reactor was calculated. These
calculated concentrations are shown below in Figure 2.
Using the constructed standard curve for phosphorus concentration shown
below in Figure 3, the concentration of phosphorus in each reactor was
calculated. These calculated concentrations are shown below in Figure 4.
The pH measurements for each reactor are shown below in Figure 5.
Figure 6. Monod model of growth of C.
protothecoides based on phosphorus
concentration and light intensity
Figure 7. Modeled concentration of C. protothecoides
in a batch reactor over time
Figure 8. Modeled concentration of phosphorus in a batch
reactor over time
Standard 1
y = 0.1021x
R² = 0.957
Standard 2
y = 0.0981x
R² = 0.9966
0
0.1
0.2
0.3
0.4
0.5
0.6
0 1 2 3 4 5 6
Absorbance
Phosphorus Concentration (mg/L)
November 30th
December 2nd &
3rd
y = -0.0431x + 18.462
R² = 1
y = -0.044x + 15.758
R² = 0.9176
y = -0.0106x + 3.8604
R² = 0.8613
0
2
4
6
8
10
12
14
16
18
60 70 80 90 100 110 120 130 140 150
CalculatedPhosphorusConcentration(P)[mg/L]
Time (t) [hr]
BG11
BG11 & Detergent
Deionized Water & Detergent
y = -0.0037x + 9.5744
R² = 0.3905
y = -0.0078x + 9.5446
R² = 0.6719
y = 0.0063x + 5.6942
R² = 0.3782
0.0
2.0
4.0
6.0
8.0
10.0
12.0
0 20 40 60 80 100 120 140
pH
Time (t) [hr]
BG11
BG11 & Detergent
Deionized Water and Detergent
0
5
10
15
20
25
30
35
40
45
0 20 40 60 80 100 120
ConcentrationofAlgae[mg/L]
Time (t) [hr]
BG11
BG11 & Detergent
Deionized Water & Detergent
Figure 2. Measured concentration of C. protothecoides
over time
Figure 1. Standard curve for the total
suspended solids and optical density for diluted
freshwater algae culture
Figure 4. Measured concentration of phosphorus over
time
Figure 3. Standard curve for phosphorus
concentration and absorbance for diluted
freshwater algae culture
Figure 5. Measured pH values over time
y = 0.0019x
R² = 0.9699
0
0.1
0.2
0.3
0.4
0.5
0.6
0 50 100 150 200 250 300
OpticalDensity(OD750nm)
Total Suspended Solids [mg/L]
![Modeled growth of C. protothecoides with Phosphorus and Light as Limiting Nutrients
Patrick Cusack and Rachael Mordovancey
Dr. Caye Drapcho
BE 4100, Biosystems Engineering,
Clemson University, Clemson, SC, 29361
Abstract
This project was performed to model a biological growth system
using the Monod model for multiple limiting substrates. Three
batch reactors inoculated with Chlorella protothecoides were
grown to measure growth in the presence of the plant-based,
phosphorus-free Dropps laundry detergent. The results showed
that C. protothecoides grew best in the absence of the detergent.
Additionally, the Monod model showed that C. protothecoides
would be able to grow exponentially for 2500 hours, after which
the phosphorous concentration would be depleted.
Introduction
In 1948, manufacturers began adding sodium tripolyphosphate
to synthetic detergents for its impressive cleaning abilities. As its
use became more popular, the concentration of phosphorus in
household wastewaters also began to rise. Eventually, this excess
phosphorus wound up in streams, lakes, and the ocean. With
such excess phosphorus, in combination with sufficient sunlight,
nitrogen, and other essential nutrients, freshwater algae
flourished. In some cases, the algae grew too well and depleted
the dissolved oxygen concentration in the water, suffocating fish
and other aquatic animals. When scientists made the connection
between excess phosphorus and eutrophication, most developed
countries banned the use of phosphates in detergents.
Unfortunately, with the widespread use of agricultural
fertilizers, eutrophication is still a prevalent environmental
concern. The purpose of this experiment was to test the effects of
the Dropps laundry detergent on the growth of C. protothecoides
and model the growth of C. protothecoides with phosphorus and
light as the substrates of interest.
Materials and Methods
Three beakers of varying phosphorus levels were inoculated
with 21 mL of C. protothecoides. The first beaker contained 700
mL of BG11 media. The second beaker contained 700 mL of
BG11 media and 540 µL of Dropps laundry detergent. The third
beaker contained 700 mL of deionized water and 540 µL of
Dropps laundry detergent. All three beakers were then placed on
stir plates. The optical density of each sample was measured at
750 nm with a spectrophotometer. The pH of each sample was
measured using a pH reader. Lastly, the phosphorus
concentration of each sample was measured following Method
4500-P D of Standard Methods for the Examination of Water
and Wastewater (APHA, 1995). Additionally, the growth of C.
protothecoides, based on phosphorus concentration and light
intensity, was modeled using the program STELLA.
Conclusions
Although the concentration of C. protothecoides increased and decreased
over time in all three reactors, the algae grew best in the BG11 without the
Dropps laundry detergent. The concentration of phosphorus in each reactor
consistently decreased, indicating phosphorus utilization and biomass
formation. Interestingly, the reactor containing deionized water and the
“phosphorus-free” Dropps detergent supported the growth of C.
protothecoides. Unless there was contamination or errors conducting the
phosphorus test, this indicates that the detergent contains phosphorus.
Unfortunately, with such a short span of time, this data does not accurately
show the effects of the Dropps detergent on C. protothecoides growth.
References
1. Köhler, J. (2016). Detergent Phosphates: An EU Policy Assessment. J. of Business Chem., 3-2.
http://www.businesschemistry.org/article/?article=73
2. Mordovancey, R. 2018: Lab 5 Methods of Biomass Determination: Optical Density and
biomass Dry Weight. BE 4100, Clemson University.
3. Drapcho 2018: Lecture 11 Stoichiometry of Microbial Reactions. Unpublished Notes, BE 4100,
Clemson University.
4. Drapcho 2018: Lecture 12 Modeling Microbial Growth with Monod Model. Unpublished
Notes, BE 4100, Clemson University.
5. Drapcho 2018: Lecture 14 Product Formation and Nutrient Utilization Rates. Unpublished
Notes, BE 4100, Clemson University.
6. SC-Life Presentation Series: How to Create a Poster for a Professional Meeting, Stocks and
Zimmerman, 2005.
Acknowledgements
We would like to thank Dr. Caye Drapcho for her expertise and assistance and the
Environmental Engineering and Earth Sciences department for the equipment and lab
space.
Results
Using the standard curve from a previous lab, shown below in Figure 1, the
concentration of C. protothecoides in each reactor was calculated. These
calculated concentrations are shown below in Figure 2.
Using the constructed standard curve for phosphorus concentration shown
below in Figure 3, the concentration of phosphorus in each reactor was
calculated. These calculated concentrations are shown below in Figure 4.
The pH measurements for each reactor are shown below in Figure 5.
Figure 6. Monod model of growth of C.
protothecoides based on phosphorus
concentration and light intensity
Figure 7. Modeled concentration of C. protothecoides
in a batch reactor over time
Figure 8. Modeled concentration of phosphorus in a batch
reactor over time
Standard 1
y = 0.1021x
R² = 0.957
Standard 2
y = 0.0981x
R² = 0.9966
0
0.1
0.2
0.3
0.4
0.5
0.6
0 1 2 3 4 5 6
Absorbance
Phosphorus Concentration (mg/L)
November 30th
December 2nd &
3rd
y = -0.0431x + 18.462
R² = 1
y = -0.044x + 15.758
R² = 0.9176
y = -0.0106x + 3.8604
R² = 0.8613
0
2
4
6
8
10
12
14
16
18
60 70 80 90 100 110 120 130 140 150
CalculatedPhosphorusConcentration(P)[mg/L]
Time (t) [hr]
BG11
BG11 & Detergent
Deionized Water & Detergent
y = -0.0037x + 9.5744
R² = 0.3905
y = -0.0078x + 9.5446
R² = 0.6719
y = 0.0063x + 5.6942
R² = 0.3782
0.0
2.0
4.0
6.0
8.0
10.0
12.0
0 20 40 60 80 100 120 140
pH
Time (t) [hr]
BG11
BG11 & Detergent
Deionized Water and Detergent
0
5
10
15
20
25
30
35
40
45
0 20 40 60 80 100 120
ConcentrationofAlgae[mg/L]
Time (t) [hr]
BG11
BG11 & Detergent
Deionized Water & Detergent
Figure 2. Measured concentration of C. protothecoides
over time
Figure 1. Standard curve for the total
suspended solids and optical density for diluted
freshwater algae culture
Figure 4. Measured concentration of phosphorus over
time
Figure 3. Standard curve for phosphorus
concentration and absorbance for diluted
freshwater algae culture
Figure 5. Measured pH values over time
y = 0.0019x
R² = 0.9699
0
0.1
0.2
0.3
0.4
0.5
0.6
0 50 100 150 200 250 300
OpticalDensity(OD750nm)
Total Suspended Solids [mg/L]](https://image.slidesharecdn.com/postertemplate-18-191121024322/85/Biological-Kinetics-1-320.jpg)
